J. Agr. Sci. Tech. (2015) Vol. 17: 765-776 Molecular Identification of an Isolate of Peanut Mottle Virus (PeMoV) in Iran ∗ N. Beikzadeh 1 , A. Hassani-Mehraban 2, and D. Peters 3 ABSTRACT Peanut plants showing mottling, yellow and necrotic spots on leaves were collected from peanut fields in Golestan province. Electron microscopic studies revealed the presence of flexuous filamentous particles ca. 700 nm in length, which was suggestive of a potyvirus infection. Healthy Nicotiana benthamiana plants mechanically inoculated with sap from infected peanut plants showed mottling, downward leaf curling, and wrinkling of the leaves. The virus was transmitted by Myzus persicae in a non-persistent manner to healthy N. benthamiana, on which symptoms were observed two weeks later. RT-PCR using an Oligo-dT and a NIb primer set resulted in a fragment of about 1093 bp, which comprised the complete coat protein (CP) gene and 3´-non-coding region. Analysis of its CP nucleotide and amino acid sequence revealed 98-99% similarity and 95-99% identity to those of Peanut mottle virus (PeMoV) isolated from other countries, respectively. The molecular data confirmed serological, vector transmission, and electron microscopic findings on the incidence of PeMoV in Iran. Additionally, sequence and phylogenetic analyses of the CP revealed clustering of Iranian PeMoV isolate with Asian/Australian isolates. Keywords : Arachis hypogea , Phylogenetic analysis, Potyviridae . INTRODUCTION Cucumber Mosaic Virus (CMV), Peanut Stripe Virus (PStV) and Peanut Stunt Virus Peanut ( Arachis hypogaea L.) is grown in (PSV), which are transmitted through seeds, tropical and temperate regions as oilseed are the most devastating viruses infecting crop in China, India, and the United States peanut crop. These viruses are of particular (Reddy, 1991). Peanut is the fourth most economic importance in developing commonly produced oilseed in the world countries as they cause severe reductions in Downloaded from jast.modares.ac.ir at 22:13 IRST on Monday September 27th 2021 after soybean, rapeseed, and cottonseed seed yield and quality (Spiegel et al ., 2008; (Ozudogru, 2011). In Iran, this crop is Akin and Sudarsono, 1997; Kuhn, 1965; Xu mainly grown in the provinces of Golestan, et al ., 1991; Xu et al ., 1998). Peanut Mottle Khouzestan, and Guilan for the production Virus (PeMoV), a species of the genus of consumption nuts (Noorhosseini Niyaki Potyvirus, occurs worldwide (Behncken, and Haghdoost Manjili, 2009; Radjabi and 1970; Sreenivasulu and Demski, 1988). This Noorhosseini Niyaki, 2010). More than 20 virus was first found to infect peanuts and viruses belonging to different virus families soybean (Glycine max ) in Georgia, USA infect peanut naturally (Spiegel et al., 2008). (Kuhn, 1965). PeMoV is transmitted by _____________________________________________________________________________ 1 Khorasan Razavi Agricultural Education Center, Technical and Vocational Higher Education Institute, Mashhad, Islamic Republic of Iran. ∗ Corresponding author; e-mail: [email protected] 2 The Netherlands Food and Consumer Product Safety Authority, Section of Virology and Molecular Biology, P. O. Box: 9102, 6700 HC Wageningen, The Netherlands. 3 Department of Plant Sciences, Laboratory of Virology, Wageningen University, P. O. Box: 629, Wageningen, The Netherlands. 765 _____________________________________________________________________ Beikzadeh et al. different aphid species like Myzus persicae, approach to confirm the presence of PeMoV Aphis craccivora, A. gossypi and in Iran. The sequences obtained from the Rhopalosiphum padi (Behncken, 1970; coat protein and 3´-non-coding region of Sreenivasulu and Demski, 1988). Its PeMoV are compared with other isolates transmission by seeds is assumed to be the described in other countries. reason for its present worldwide distribution in peanut (Dang et al ., 2010; Gillaspie et al ., MATERIALS AND METHODS 2000). The virus has also been found in other important crops, such as pea ( Pisum sativum ) and bean ( Phaseolus vulgaris ) Virus Source (Brunt et al ., 1990). Two viral diseases in peanut crop have In June 2009, peanut plants showing been reported in Golestan province. Their mottling and yellow and necrotic spots symptoms resemble those earlier described (Figure 1) were collected from five peanut for a disease assumed to be caused by fields in Golestan province. Sap from PeMoV (Elahinia et al ., 2008; Shahraeen infected leaf samples was mechanically and Bananej, 1995), but also a tospovirus inoculated on Petunia hybrida (as an Groundnut Bud Necrosis Virus (GBNV) indicator plant for tospovirus infections) (Golnaraghi et al ., 2002). Since their first using 0.5M phosphate buffered saline (PBS), discovery, similar symptoms have been pH 7.0., containing 0.01% Na 2SO 3 (Allen frequently observed in peanut fields in the and Matteoni, 1991; de Ávila et al ., 1993) same area. The disease on peanut was and Nicotiana benthamiana to maintain the highlighted by mottling, necrosis on the virus isolates, and stored at -80°C for further leaves and necrotic areas on the shoots. studies. Although initial detection of both viruses in Iran was mainly based on enzyme-linked immunosorbent assay (ELISA), little is Host Range Study known about their molecular identity. Here, we present molecular data of the viral agent In addition to P. hybrida and N. of the disease using reverse transcriptase benthamiana , crude extracts of infected polymerase chain reaction (RT-PCR) peanut leaves were inoculated to a limited Downloaded from jast.modares.ac.ir at 22:13 IRST on Monday September 27th 2021 Figure 1. Peanut leaves and a plant showing symptoms of PeMoV. Chlorotic areas progressing to necrotic spots on the leaves and stem are shown. 766 Molecular Identification of a PeMoV Isolate _____________________________________ number of plant species including transcription polymerase chain reaction (RT- Chenopodium quinoa, Chrysanthemum sp., PCR) assay for the presence of virus. Emilia sonchifolia, Cucumis sativus, and Capsicum annuum . Electron Microscopy Serological Tests Extracts of N. benthamiana plants with systemic mottling and downward curling of Polyclonal antisera (IgG and conjugate) the leaves and healthy plants were used to raised against GBNV and PeMoV were prepare leaf dip preparations. They were applied to detect these viruses in the negatively stained with 2% uranyl acetate collected leaf samples and in inoculated N. (pH 3.8) and examined with a JEOL benthamiana plants using the double JME1011 electron microscope. antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Clark Total RNA Extraction and RT-PCR and Adams, 1977) The PeMoV serum and positive control material were kindly provided by Dr. S. Winter, Leibniz Institute To confirm the identification of the virus DSMZ, Braunschweig, Germany. The found in the samples with positive PeMoV GBNV reagents were used in a dilution of 1 reactions in DAS-ELISA, total RNA was µg mL -1 and the PeMoV reagents in a extracted from 100 mg healthy and infected dilution of 2 µg mL -1. The absorbance fresh N. benthamiana leaf tissue using Trizol values were measured at 405 nm by an reagent according to manufacturer’s ELISA-reader (FLUOstar OPTIMA (BMG instruction (Invitrogen, USA). A primer set LABTECH GmbH, Germany) after 60 was applied to target the 3´-non-coding minutes. Samples with absorbance values region (3´-NCR) sequence and coat protein greater than or equal to three times the gene (Figure 2-B). The first cDNA strand average of negative control were considered was synthesized from the purified RNA with to be positive. Potyvirid Oligo-dT (17) [5'- CACGGATCCCGGG(T)17VGC-3' complementary to the 3’terminal poly-A of Aphid Transmission Test potyvirid species with an additional Bam HI (underlined) sequence] and GBNV-R [5'- Five-leaf-stage N. benthamiana plants TTACAATTCCAGCGAAGGAC-3' at Downloaded from jast.modares.ac.ir at 22:13 IRST on Monday September 27th 2021 were used as test plants. Virus-free green nucleotide position 2160-2180 based on the peach aphids adults, Myzus persicae Sulz., entire sequence of the S RNA segment (Acc. reared on healthy radish plants, were starved No. U27809)] (Gibbs and Mackenzie, 1997; for 1 h and transferred onto infected N. Satyanarayana et al. , 1996, Zheng et al ., benthamiana plants for an acquisition access 2010) using AMV-reverse transcriptase feeding period of 2-5 minutes. Groups of 20 (Promega, USA) and incubated for 1 h at aphids were transferred to two healthy N. 42ºC. To amplify the PeMoV coat protein benthamiana plants. Aphids were removed and GBNV N genes, a forward primer after an inoculation access period of 1-2 annealing to nuclear inclusion B (NIb) gene hours. As negative control, virus-free aphids (NIbFor: 5'- were placed on healthy N. benthamiana TGATGAAGTTCGTTACCAGTC-3') leaves and transferred to healthy plants identical to nucleotide position 8567-8587 (Sreenivasulu and Demski, 1988; Samad et and GBNV-F (5'- al ., 1993). The plants were kept for 2 weeks ATGTCTAACGTCAAGCAAC-3') to in the greenhouse for symptom appearance nucleotide position 2971-2990 were and then analyzed using reverse- designed based on the alignment of two NIb 767 _____________________________________________________________________ Beikzadeh et al. sequences from PeMoV isolates (Acc. Nos. RESULTS AF023848 and X73422) and the start codon of the N gene, respectively. PCR- Host Range and Serology amplification for both viruses was carried out using GoTaq polymerase (Promega) under the conditions: